| Inflammatory reaction refers to the defense response of the living tissue with the vascular system to the damage factor,and is an important defense mechanism for the body to resist external stimuli.Inflammation has the proper immune enhancing effects on the body.When the content of inflammatory cytokines than body self-defense system clearance ability or a long time when the body is stimulated,can occur cascades caused by acute and chronic inflammation.Severe form of systemic inflammatory response syndrome(SIRS)patients with life-threatening.The current research suggests that lipopolysaccharid is the main cause of SIRS.LPS is the main effector cells of monocyte macrophage system.When the effect of LPS on the cell,and it is binding on receptor TLR4.After signaling complex transfer,activities Nuclear factor-?B(NF-?B)and activator protein 1(AP-1).The transcription and expression regulation of intracellular inflammation related genes,which mediated inflammation.At present,the inflammatory reaction induced by LPS,mainly used antibiotics such as polymyxin B,hydrocortisone,dexamethasone and budesonide and other drugs for treatment.However,the long-term use of these drugs can cause many adverse reactions,such as reducing immune function,increasing infection and increasing damage to kidneys,which greatly limits their clinical application.Therefore,screening anti-inflammatory effects from natural products,low toxicity and extensive sources of anti-inflammatory drugs have gradually become the main direction of new drug research and development.Quercetin is a kind of flavonoids.It is a traditional Chinese medicine,which is commonly found in the flowers,leaves and fruits of plants.AMP-activated protein kinase(AMPK)is an evolutionarily conserved serine / threonine protein kinase,known as the "cell energy regulator",which plays an important role in maintaining cell and body energy balance.In recent years,it has been found that the activity of AMPK is closely related to the inflammatory reaction.The results of previous studies have shown that quercetin has a significant therapeutic effect on LPS induced SIRS model in mice.And it can activate AMPKin many adipocytes to play the role of metabolic regulation.Therefore,the inhibition mechanism of quercetin on inflammation and its relationship with AMPK were studied by using LPS to induce the cell model of RAW264.7 as an inflammatory reaction.The specific results are as follows:1.The cytotoxicity of quercetin was investigated by detecting the effect of quercetin on the proliferation of RAW264.7 cells at different concentrations.The results showed that 5-20?M quercetin macrophages after 24 h,compared with the control group,the cell survival rate were 98.9%,96.7% and 95.5%.It is showed that quercetin in the concentration range of 5-20?M in the activity of RAW264.7 cells had no significant effect(P > 0.05).In this paper,5,10 and 20?M concentration of quercetin was used for subsequent experiments.2.The effects of quercetin on the secretion of TNF-?,PGE2 and NO induced by LPS induced RAW264.7 cells were detected,to investigat the inhibitory effect of quercetin on the inflammatory response.The results showed that quercetin could significantly inhibit the secretion of TNF-?,PGE2 and NO in RAW264.7 cells.Different concentrations of quercetin(5,10,20 ?M)interfered significantly in the secretion of TNF-?,PGE2 and NO compared with LPS group(P < 0.01),and showed a dose-dependent manner.The inhibitory rates of 20?M quercetin on TNF-?,PGE2 and NO were 77%,68.2% and 52.9%,respectively(P < 0.01).It shows that quercetin has significant anti-inflammatory effects.3.The effect of quercetin on the expression of TNF-?,COX-2 and iNOS genes in LPS induced RAW264.7 cells was examined,to investigat the mechanism of quercetin inhibiting the inflammatory response at the gene level.The results showed that quercetin could inhibit the mRNA expression of TNF-?,i NOS,and COX-2 in LPS induced RAW264.7 cells to varying degrees.The 20 ?M quercetin,compared with group LPS,inhibition of the expression of TNF-?,iNOS and COX-2 m RNA rates were 63.37%,60.38% and 54.06%(P <0.01).It shows that quercetin can inhibit the expression of pro-inflammatory factors to play an anti-inflammatory effect.4.The effect of quercetin on the expression of NF-?B p-p65,c-jun and c-fos in LPS induced RAW264.7 cells was examined,to investigat the mechanism of quercetin inhibiting inflammatory response at the protein level.The results showed that quercetin could inhibit the protein level of the transcription factor NF-?B p-p65,c-jun and c-fos in LPS induced RAW264.7 cells in a dose-dependent manner.The 20 ?M quercetin,compared with the LPS group,inhibition of the expression of NF-?B p-p65,c-jun and c-fos protein were 76.77%,73.14% and 70.62%(P < 0.01).Quercetin can play an anti-inflammatory effect by inhibiting the protein expression of transcription factors.5.The effects of quercetin on the phosphorylation of AMPK protein in RAW264.7 cells induced by LPS were detected,to investigat the activation of quercetin to AMPK.The resultsshowed that quercetin could increase the phosphorylation level of AMPK protein in RAW264.7 cells induced by LPS.5,10 and 20 ?M quercetin,compared with the LPS group p-AMPK ?1 protein expression was increased by 1.82 times,2.25 times and 2.92 times(P <0.01).It is deduced that the anti-inflammatory effect of quercetin is related to the activation of the AMPK pathway.6.To investigate the effect of AMPK activator,inhibitor and quercetin on the expression level of NF-?B p-p65,c-jun and c-fos in LPS induced RAW264.7 cells,to investigat the mechanism of quercetin inhibiting inflammatory response and the relationship between AMPK.The results showed that AMPK activator and quercetin significantly decreased the protein expression level of NF-?B p-p65,c-jun and c-fos in LPS induced RAW264.7 cells(P< 0.01),indicating that quercetin could play an anti-inflammatory role by activating phosphorylation level of AMPK protein. |
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